A study of redox-signalling through generation of redox related biosensors

During high light, plants are subject to elevated levels of ROS induced by electron flow at photosystems I and II and from photorespiration. The main ROS, H2O2 is reduced by peroxidases, which obtain their reducing power from antioxidants such as ascorbate and glutathione. Ascorbate and glutathione in turn get reduced by NADH and NADPH produced by respiration or ultimately photosynthesis. These levels of H¬2O2 and the resulting change in cellular redox state yields signals for plants to drive signalling responses to biotic and abiotic stresses, development, growth and cell death. H¬2O2 was found to have a role in systemic signalling and was shown to accumulate in bundle sheath cells during stress. H2O2 can be monitored by the ratiometric fluorescent probes roGFP2-Orp1 and HyPer, although the latter is subject to posttranslational gene silencing in transgenic plants. Furthermore, HyPer needs to be corrected for pH sensitivity of the probe. The ratiometric fluorescent probe GRX1-roGFP2 can monitor the redox state of gluthatione. These genetically encoded sensors can be expressed in different tissues and subcellular localisations. In this study, biosensors to monitor H¬2O2 and glutathione redox potential (EGSH) in cytosol and chloroplast stroma in bundle sheath cells were developed using Golden Gate cloning. These four constructs were successfully transformed into Arabidopsis thaliana, which – once homozygous – can be used to monitor systemic stress signalling during experiments. SypHer, a H2O2 insensitive version was created out of HyPer using site-directed mutagenesis. This was to correct for pH effects of HyPer in ten subcellular compartments. Using confocal laser scanning microscopy, this pH-probe has shown to produce relative pH quantifications in subcellular compartments similar to that reported in the literature. However, a calibration curve is essential to be able to calculate absolute pH values. The generation of a meaningful calibration curve did not succeed in this study, hence no hard numbers could be generated. However, suggestions to improve the method to generate a pH calibration curve were made. Nevertheless, the relative pH data can be used to correct for earlier acquired H2O2 data measured in the same subcellular localisations with HyPer.